Field of the Invention
The present invention relates generally to screening assays and more particularly to a method, system and composition for screening agents that affect calcium flux, membrane depolarization, and/or the propagation of the action potential in cardiomyocytes.
Background Information
Cardiac arrhythmia and sudden cardiac death are two of the major topics for pharmaceutical industry both from therapeutic and toxicologic views. Atrial (AF) and ventricular (VF) fibrillation are the most common forms of arrhythmia and have major clinical implications. AF is the most prevalent in the population and its occurrence arises in the aging population, leading to the estimation that more than 15 millions of Americans will be affected by 2050. AF is also the major cause of stroke in elderly and is associated particularly with the more severe forms. The underlying cause of cardiac arrhythmia is the abnormal impulse formation and propagation in the heart resulting from altered (overactive or underactive) function of ion channels and exchangers that ultimately lead to an alteration of the heart beat rate and intensity of contraction. Therefore, arrhythmia undermines the process whereby electrical excitation leads to the muscular contraction that provides the pumping function of the heart.
A new generation of anti-arrhythmic drugs for example is under development to target channels that are specifically expressed in the atria, like Kv1.5 potassium channel that is responsible for the IKur current. Abnormalities in intracellular Ca2+ handling occur in a range of arrhythmogenic conditions, including congestive heart failure (CHF), ischemic heart disease, myocardial hypertrophy and inherited diseases such as catecholaminergic polymorphic ventricular tachycardia (CPVT). Calcium leak from the SR or attenuated calcium uptake are known to promote after-depolarizations that lead to atrial and ventricular fibrillation. Modulating the interaction between RyR2 receptor and the Calstabin 2 regulator is one of the approaches under evaluation to reduce Ca2+ leak in patients with chronic AF.
Another target for future anti-arrhythmic drugs is the intracellular coupling machinery. Gap junctions are clusters of closely packed hemichannel subunits that connect adjacent cells to allow the passage of ions and small molecules end electrically coupling the nearby cardiomyocytes in the heart. Connexins 43, 40 and 45 are the most abundantly expressed in myocardium, where connexin 43 dominates working ventricular and atrial myocytes while connexin 40 is expressed in atrium and along with connexin 45 in conduction tissue. Changes in gap junction function and organization has been identified in several arrhythmia models and is known to affect the conduction velocity. Increasing gap-junctional coupling can favor anti-arrhythmia by improving cell-to-cell communication and thereby conduction velocity. Anti-arrhythmic peptides were first identified in 1980 and led to the identification of the stable analogue rotigaptide.
Another concern for cardiac arrhythmia is represented by new drugs that are under development or have been approved recently for marketing, when their effects in a large sample population have not been completely evaluated. In recent years several blockbuster drugs were withdrawn from the market or have undergone severe labeling restrictions due to safety issues. The widely popular non-sedating antihistamine Terfenadine (Seldane™), the gastric prokinetic Cisapride (Propulsid™) and the blockbuster anti-inflammatory Rofecoxib (Vioxx™) are examples of drugs removed from the market for causing polymorphic ventricular tachycardia (torsade de pointes or TdT) and sudden cardiac death. Cardiotoxicity is also a substantial problem in the field of anti-cancer drugs, as effective anti-neoplastic agents in the anthracycline class (e.g., doxorubicin) are cardiotoxic likely by interfering with cardiac metabolism. Thus, there is an ongoing effort to identify compounds that will be effective anti-tumor agents but not affect heart muscle cells, or to develop cardioprotective agents to be administered with the anthracycline-class compounds.
Drugs that have been withdrawn aftermarket are only the tip of the iceberg, although they are the most stunning examples, and it has been estimated that between 80% and 96% of the drugs under development fail the clinical trials, mainly for toxicity concerns. Withdrawal of medications from late stage development and from the market can cause severe financial losses, considering that the cost of a new drug development has been estimated in the range of up to $1.6 billion.
The main target of drug cardiotoxicity is the human homolog of Drosophila's EAG gene (hERG), a K+ channel with properties resembling those of the IKr current. Blockade of the hERG channel is known to delay the repolarization current and induces a prolongation of the ventricular action potential and increases the possibility of early after-depolarization (EAD), resulting from reopening of L-type calcium channels during the late repolarization phase. Furthermore, a reduction in hERG current is known to have a greater effect on mid-myocardium relative to other regions of ventricular walls increasing the heterogeneity of tissue refractoriness. These two alterations increase the risk of arrhythmia and numerous evidences suggest that several non-cardiac drugs bind to hERG channels, increasing the risk of QT prolongation and torsades de pointes (TdT), a specific variety of ventricular tachycardia characterized by a distinctive twisting of the QRS complex around the baseline in the ECG and at high risk of degenerating into ventricular fibrillation and sudden death.
A remarkable feature of compounds that have been associated with high risk of TdT is their large diversity in both therapeutic action and chemical structure. Different from other channels, hERG has a large inner cavity size where the compounds tend to be trapped on closure of the activation gate. The inhibition is contingent upon channel gating; suggesting that the channel needs to open for compound binding, with consequent relatively slow recovery after washout.
Despite the multiplicity of causes for arrhythmia (environmental, age, genetic background) and the fact that arrhythmia targets specific regions of the heart (AF vs. VF), with differences in impulse generation and propagation, there are only few anti-arrhythmic drugs with low selectivity and their use has decreased in the past years. This is mainly due to problems with the ability of the drugs to have a larger pro-arrhythmic effect in other regions of the heart. There is therefore a major unmet need for drugs that will control arrhythmia more safely and effectively.